EP1832874A1 - Substrate surface with hydrophobic and hydrophilic regions - Google Patents

Abstract

Modifying a hydrophilic or hydrophobic support surface so that biomolecules can be immobilized within discrete detection zones comprises coating the surface with a hydrophobic layer and selectively removing the hydrophobic layer. If the support surface is hydrophobic, it then converted to a hydrophilic surface. Independent claims are also included for: (1) analytical or diagnostic device with a surface produced by a method as above in which the detection zones are treated with a solution of crosslinkable or polymerizable components in a hydrophilic solvent and incubated so that the components form a crosslinked or polymerized layer with hydrophilic properties; (2) analytical or diagnostic device with a surface having both hydrophilic and hydrophobic regions, where the hydrophilic regions serve as detection zones and have hydrophilic guide structures.

Description

The present invention relates to the analysis and diagnosis of biomolecules such as nucleic acids and polypeptides. In particular, the invention relates to a method of modifying carrier surfaces and devices comprising these modified surfaces.

In the field of solid phase assisted biomolecule diagnostics, numerous techniques have been established using different solid phase systems, with the support surfaces of these conventional systems being essentially constructed of plastic, glass, silica glass, quartz, or silicon.

It will be apparent to those skilled in the art that using these solid-phase systems, devices capable of simultaneously analyzing or simultaneously detecting a plurality of different or the same biomolecules can be prepared. Such devices are known in the art and are referred to, for example, as biochips, biosensors or microarrays. More recent developments are based on electronic semiconductor chips (microchips) with functionalized surfaces, for example for the covalent bonding of biomolecules for analytical and / or diagnostic applications and measuring methods. A common feature of these devices is that they are based on the specific interaction between solid phase-coupled receptor molecules and ligands to be detected of a sample to be tested, which specific interactions result in the formation of receptor / ligand complexes whose presence or absence can be detected by appropriate techniques ,

The biomolecules are printed on activated surfaces of the different carrier systems for the production of so-called microarrays. For this purpose, the biomolecules are applied to the surface in mostly aqueous solutions and bound to the surface via chemical and physical processes. The application is usually carried out using contactless printing techniques using e.g. Inkjet printers. Alternatively, contact systems using e.g. Dot matrix printers used. Common to these techniques is that small droplets (spots) are applied to the surface with them.

In order to achieve high sensitivities and / or specificities and at the same time to enable the simultaneous execution of a multiplicity of parallel reactions in multiplex format on the smallest possible surface, enormous efforts have been made in the prior art, although not all problems could be solved.

For sensitive surfaces such as polymer gels, in most cases only contactless printing techniques can be used (eg inkjet printers). This often leads to the problem that different media in the nozzles of the printer show different printing behavior and thus lead to different printing results, through which the results of eg a comparative multiplexing application can be falsified or rendered useless. In particular, when printing sensor surfaces with a plurality of grid-like detection points or fields, it depends on the dosage and positioning accuracy. The morphology, size and volume of the applied spots should be identical. Furthermore, each printed spot should ideally be located on the surface to ensure optimum measurement or detection. For example, if spots are printed next to the halftone dots or not centered exactly on them is clear that the measurement results can not be reproduced. In extreme cases, in the case of imprecise printers and superhydrophobic surfaces, blending of the spots may occur because the adhesion of droplets to smooth and water-repellent surfaces is very low. In the case of very hydrophilic or absorbent surfaces with good wettability, an uncontrolled spread of the droplet can occur, so that the morphology and positional accuracy of the droplet are also at risk here.

In printing biomolecules onto patterned silicon substrates, it is observed that the coating and patterning of the semiconductor surface can have a major impact on the wettability of the substrate and the contact angle of a printed spot. With biosensors in the form of microchips, a very high dosing and positioning accuracy must be required so that the topography of the microelectronics optimally match the array design and the printing result. Even the slightest inaccuracies in positioning the spots can jeopardize reproducible results. In addition, the ratio between the pressure volume and the predetermined wetting area of the detection point or area should be set so that as far as possible the entire detection area is provided with sufficient liquid in order to ensure drying effects such as e.g. the so-called donut effect play no role or can be neglected.

The problem underlying the present invention is therefore to provide optimized surfaces or to provide suitable production processes for the same in the field of biosensors, which overcome the problems of the prior art with regard to the positioning accuracy of the printed spots.

The object is achieved by providing a method according to the main claim. Preferred embodiments of the method according to the invention are set forth in the subclaims and in the following description of the invention. Furthermore, the object is achieved by providing devices which have the surfaces modified according to the invention.

Using a surface modified according to the invention, the positioning accuracy of the spots with respect to the predetermined grid patterning of a semiconductor surface could be optimized in an impressive manner. Even spots with a diameter of about 50 microns were after printing surfaces of the invention exactly in the center of their intended detection points or areas, whereby the sensitivity of appropriately equipped systems and the reproducibility of the results obtained with them over conventional systems could be significantly increased , By virtue of the improved properties of the surface according to the invention, the actual spot size at an exemplary print volume of 200 nl of an aqueous protein mixture, e.g. be reduced to a diameter of about 0.2 mm, wherein the edge regions of the applied drop were formed discretely. Using different print volumes, further results showed that the diameter of an applied spot can be adjusted according to the invention between 0.02 and 1.0 mm, with a diameter between 0.05 and 0.250 mm being particularly preferred.

The invention is based on the recognition that the surface of the biosensor must be divided into two functional areas with respect to their properties before the immobilization of the receptor molecules in order to be able to realize the above-mentioned optimizations. As usual biochips or Sensors in the format of a grid-like array discrete detection areas or detection points or fields have, in which the formation of detectable complexes of receptors and ligands can be expected, the total area of these detection areas is the first functional area, while the second functional area the remaining Area comprises and excludes the detection areas according to the invention.

According to the invention, the properties desired for the two functional regions relate to the wettability or swellability of the reaction surface when using aqueous systems which contain biomolecules. It is known that hydrophobic surfaces impede the attachment / absorption of biomolecules applied in aqueous medium, whereas this is favored by hydrophilic surfaces. It follows according to the invention that the detection areas have hydrophilic, but the remaining area hydrophobic or superhydrophobic properties.

It is known in the art, for example, that the contact angle of a drop of liquid on the surface of a solid basically depends on the interaction between the substances at the contact surface. The smaller this interaction, the larger the contact angle becomes. In the special case according to the invention of the use of water or aqueous pressure volumes, the surface is called hydrophilic at low contact angle (about 0 °), hydrophobic at angles of 90 ° and superhydrophobic at even larger angles. The latter is also called the lotus effect at very high angles (about 160 °) and corresponds to an extremely low wettability.

Accordingly, a desired result for the first functional area (total area of the detection areas) Hydrophilicity, which is characterized by the use of aqueous systems by a contact angle of about 0 to about 90 °, preferably by a contact angle of 0 to 70 °. It follows that the second functional region (residual surface without detection regions) has a desired hydrophobicity which, when aqueous systems are used, is characterized by a contact angle of about 70 to about 170 °, preferably by a contact angle of 90 to 160 °

According to the invention, the geometry of the detection points or fields in the first functional region is not only round, oval or rectangular, as is the case with conventional products of this kind, but designed in a star shape. Through this form of detection points or fields, hydrophilic conductive structures are provided, with the aid of which imprecisely deposited drops are conducted into the center of the detection areas. The exact dimensions of the star-shaped guide structures can be easily determined by the skilled person depending on the task. It is only important that the width of the guide beams from their tip to the actual detection point or field is always smaller than the contact surface of the drop. The preferred number of the guide beams, whose respective length from the tip to the periphery of the detection point or field is preferably between 50 and 1000 microns, and more preferably between 50 and 250 microns, is at least 4, with a respective number of 16 to 20 guiding rays particularly is preferred.

FIG. 1 shows the design of a biochip according to the invention. The rectangular geometry of the chip surface (1) comprises two functional areas, namely the first functional area (total area of the detection areas, each detection area (2) comprising a center for the actual detection (3) and guide structures (4)), and the second functional area (5) (residual area excluding the detection areas).

With the aid of this embodiment of the first functional region according to the invention, the surface energy is used to guide or center the drop (6) in the case of inaccurate positioning via the guide structures (4) into the detection center (3). Optically usable positioning aids such as e.g. Registration crosses (7) can be present.

The method according to the invention for the modification of commercially available carrier surfaces on which biomolecules are to be immobilized within discrete detection areas, provides that the entire carrier surface, e.g. a corresponding biosensor is divided into two functional areas, these areas differ when using aqueous systems by different wettability or swellability and thus by different contact angle of a liquid applied to the solid from each other. In other words, the above contact angles are selectively changed by the different treatment of these two functional surface areas by the method according to the invention.

Methods of modifying surfaces using an exposure mask are known in the art, e.g. from semiconductor technology (e.g., masking a board prior to an etching step).

An example of the prior art modification of surfaces relates to the publication of Chrisey et al. (Fabrication of Patterned DNA Surfaces; Nucleic Acids Research, 1996 Vol. 24, 3040-3047 ), the two photolithographic methods for the formation of screened DNA surfaces describe. A surface bearing hydroxyl groups (eg SiO ₂ ) is treated with a photochemically unstable silane layer, which spontaneously forms a self-assembled monolayer (SAM) under certain conditions. This SAM represents a template to immobilize biomolecules. Irradiation of these surfaces with UV light (193 nm) produces screened chemically reactive groups, which then react with heterobifunctional crosslinker molecules. The covalent attachment of modified synthetic DNA oligomers with a crosslinker results in a stable DNA pattern. In an alternative method, a silane film is modified with heterobifunctional crosslinker molecules and then masked with a light-insensitive putty. After exposure, the layer modified with the crosslinker is exposed and contacted with a chemically modified DNA. In this way, different DNA oligomers can be immobilized on a surface.

The photolithographic process described there as an example of a selective surface modification leads to an altered property of the photoreactive molecules in the detection or reaction region range by suitable irradiation, while the residual surface covered by the mask does not undergo activation. This technique can be applied analogously with the proviso that, according to the invention, the detection areas, but not the functional residual areas, are exposed. It follows that the selective exposure using this technology is such that only the residual functional area is masked so that there is no permanent modification by the exposure at these locations. Alternatively, this selective surface activation or modification may also be carried out using suitable radiation means, optionally under additional use of a mask or the like.

1. (a) Beaufschlagen der Trägeroberfläche mit einer hydrophoben Schicht;
2. (b) Schaffung eines oder mehrerer Nachweispunkte- oder -felder mit hydrophilen Eigenschaften, indem man
   • (i) die in Schritt (a) aufgebrachte hydrophobe Schicht im Falle einer ursprünglich hydrophilen Trägeroberfläche selektiv entfernt, oder
   • (ii) die in Schritt (a) aufgebrachte hydrophobe Schicht im Falle einer ursprünglich hydrophoben Trägeroberfläche selektiv entfernt und die ursprünglich hydrophobe Trägeroberfläche in eine hydrophile Oberfläche umwandelt.

The method according to the invention for modifying a carrier surface on which biomolecules are to be immobilized within discrete detection points or fields comprises the following steps:

1. (a) impinging the support surface with a hydrophobic layer;
2. (b) creating one or more detection points or fields with hydrophilic properties by
   • (i) the hydrophobic layer applied in step (a) is selectively removed in the case of an originally hydrophilic carrier surface, or
   • (ii) the hydrophobic layer applied in step (a) is selectively removed in the case of an originally hydrophobic carrier surface and the originally hydrophobic carrier surface is converted into a hydrophilic surface.

Accordingly, the modification according to the invention is applicable to both hydrophilic and hydrophobic support surfaces.

In the case of the desired modification of a hydrophilic carrier surface, such as a silicon or glass surface, this is applied in a first step with a hydrophobic layer. For this purpose, suitable techniques of the prior art such as dipcoating or spin coating can be used. This treatment step preferably leads to a permanent, lacquer-like and hydrophobic or superhydrophobic cover layer with a preferred layer thickness of 3 to 5 μm.

Subsequently, the selective removal of this cover layer takes place in the regions of the total surface, by which the one or more detection points or fields are defined in their spatial configuration (detection center with guide structures). The previously applied hydrophobic cover layer may e.g. be removed by suitable selective irradiation. If the topcoat is e.g. provided in the form of a thin polyimide layer, exposure of the underlying hydrophilic support surface can be accomplished by selective irradiation with UV light (e.g., 200 nm). After thorough cleaning (e.g., washing), the modified support surface can be stored, optionally after immobilization of desired biomolecules, until later analytical or diagnostic use. Alternatively, by known methods, another hydrophilic layer, such as e.g. a coupling matrix is supported and fixed. If this matrix is again to be polymerized and / or crosslinked by means of suitable irradiation and fixed on the surface, selective irradiation can be dispensed with, since the aqueous solution can be applied only to the hydrophilic detection areas and solidified there.

In connection with the use of a polyimide layer, it is pointed out that, for example, a printing device configured with optical means for improving the positioning of a printing drop on a chip grid can benefit from the surface having different colors (brown corresponds to the functional residual area, not brown corresponds to the functional detection area) , Further advantageous is, for example, the application of further optical positioning aids in the form of, for example, registration crosses, etc. Accordingly, it is preferred according to the invention that the hydrophilic and hydrophobic regions differ from one another with respect to their coloration.

In the case of the desired modification of a hydrophobic carrier surface, such as, for example, of a plastic, in a first step, the surface is also exposed to a hydrophobic, preferably superhydrophobic, layer. For this purpose, the known technology of plasma polymerization is preferably used, wherein preferably a fluoropolymer layer is produced. The selective exposure and conversion of the underlying, originally hydrophobic support surface into a hydrophilic surface may preferably be effected by plasma etching. The technology of plasma etching is known and represents a gas-chemical process, which is used industrially, for example, in semiconductor technology, in microstructure technology and in display technology. The processes caused by the plasma etching lead to a removal of the hydrophobic cover layer and at the same time to a hydrophilization of the originally hydrophobic support surface (eg oxygen plasma). After thorough cleaning, these modified carrier surfaces according to the invention can be stored until later use. However, the biomolecules are particularly preferably applied immediately after the plasma etching, because the reactive groups formed by the etching on the binding surface can advantageously be used for the covalent immobilization of the biomolecules. Alternatively, the surface is provided after the plasma etching with a hydrophilic polymer layer (see above), which may be formed for example in the form of a coupling matrix for receiving the proposed biomolecules. Selective irradiation in the case of a photocrosslinkable and / or photopolymerizable polymer layer can be dispensed with since this layer can be created and fixed only on the exposed and hydrophilically designed detection points or fields.

Thus, the carrier surfaces modified according to the invention have both a hydrophilic (first functional region) and a hydrophobic or superhydrophobic (second functional region) region. These two areas are clearly distinguished because of their different properties for aqueous systems. While the total area of the detection points or fields has good wettability, the residual area with water is difficult or impossible to wet.

The process according to the invention thus leads to products having the properties required according to the invention. These products in the form of analytical or diagnostic devices therefore have two functionally and structurally different regions, which are different in wettability. The different properties relative to aqueous systems given at the interface between detection area and residual area cause an imprecisely printed drop from the hydrophobic layer to be conducted completely via the guide structures into the center of the hydrophilic detection area.

In the case of using a mask, the design thereof is in accordance with the design of the biochip or biosensor, thereby ensuring that the exposure reaches only those areas of the surface that are not intended for the detection. The outer dimensions of the mask are obvious from the size of the diagnostic device. According to the invention, the diameter of the portions of the mask which shield the light from the residual surface corresponds to the dimension of or a detection region.

In principle, a device to be used functionally as a mask according to the invention comprises two regions. During the an area selected from a material which passes the light needed for photocrosslinking and thus induces crosslinking of the photoactivatable components at the desired location, the other area is made of a material which shields the rays from the area not to be irradiated. For example, the mask can be made of a UV-transparent material, wherein the shielding areas are additionally equipped with a suitable UV filter. Alternatively, for example, a quartz glass may be used in which the shielding regions are provided by the presence of a lacquer or metal layer impermeable to the irradiation light.

The hydrophilicity of the optionally additionally desired polymer layer (e.g., coupling matrix) results from the use of water-soluble monomers or comonomers in their preparation. Solutions for these purposes usually consist of polymers or copolymers (polyethersulfone, polyacrylonitrile, cellulose acetate or thin layers of silanes on a polymer support) and are usually prepared by spin coating or dipcoating.

In a preferred embodiment, the hydrophilic layer may be provided in the form of a self-supporting film. Such a water-swellable layer is eg in EP-A 1 202 062 described.

The preferably crosslinkable by means of light of a suitable wavelength, by means of plasma or heating and polymerizable components of the proposed solution give a thus modified surface according to the invention, depending on the selection of suitable components, the desired hydrophobic or hydrophilic properties. The term "crosslinking" as used herein means the preparation of a covalent bond of at least two Polymer structures using reactive groups. Crosslinkable are components which upon activation (eg by light) undergo a reaction with other crosslinkable components, which is why at least one of the two components has a reactive group. Crosslinkable components can also be crosslinked by an additional crosslinking agent present in the solution.

By polymerization, the invention is understood to mean the construction of a sequence of monomers or comonomers to form longer-chain, optionally branched and / or crosslinked molecular structures. Preferred polymers are macromolecules with a minimum of 100 units (monomers / comonomers). Polymerizable are components (mono- or copolymers) which, upon activation of an initiator (individually in solution or as a constituent of a polymer) are excited by the radicals formed thereby for radical polymerization.

For the production of the hydrophobic layers proposed according to the invention, in the prior art e.g. Olefins or vinyl-containing compounds are known which can be used according to the invention. Preferably, polymers consisting of vinylpyrrolidone and vinyl chloride and mixtures thereof are selected. Other preferred compounds are polymers of styrene, alkyl esters of acrylic acid and methacrylic acid (e.g., methyl methacrylic acid, glycidyl methacrylate), as well as methacrylates and acrylates having fluorinated side chains.

The solution consisting of monomers, copolymers and / or polymers, which are preferably fluorinated, either a crosslinker is added or alternatively, the copolymers contain a crosslinker as a comonomer.

With regard to the composition and use and preparation of the inventively suitable components and crosslinkers is in addition to the EP 1 202 062 A1 directed.

In the case of the invention possible activation of these initiators / crosslinkers by means of UV light, in particular aromatic ketones such as benzoin, benzil or benzophenones and derivatives thereof come into consideration. Also, advantageously, sulfur-containing aromatic ketones can be used, as long as e.g. the shift of the wavelength required for exposure in the direction of a longer wavelength range is desired. These or other initiators may also be included in the above polymerization solution.

As photocrosslinkers photoreactive groups according to the invention from the group consisting of aryl azides, benzophenones, benzothiones, anthraquinones, anthrathions, thymidine, and mixtures thereof are selected, preferably benzophenones are used.

Subsequently, the treated surface is subjected to washing to wash away the removed components from the irradiated detection areas of the surface. For this purpose, suitable conventional solvents such as H ₂ O, ethanol, toluene, etc. can be used.

Suitable biomolecules which can be immobilized on the membrane within the detection regions are proteins, peptides, polysaccharides, nucleic acids, and derivatives thereof.

Due to the use of a corresponding exposure mask or the application of a selective irradiation with the aid of suitable radiation sources, it is possible, if desired, to generate screens with a respective spot size well below 1 mm, which are suitable for the immobilization of biomolecules are. Even diameters of up to 0.01 mm can be achieved according to the invention, with a diameter of about 0.1 to 0.2 mm being particularly preferred.

With this method surface structures in the micrometer range can be generated, whereby a multiplicity of signals can be generated in parallel, which allows an optimization of automated analysis methods. Various known techniques can be used for the detection of successfully immobilized biomolecules.

According to the invention, modified surfaces or carrier surfaces are thus provided which allow the generation of very small, discrete detection areas and therefore printing with spots of a size which are far below the conventionally achievable diameter. A further advantage of the present invention is that correspondingly modified surfaces permit virtually no unspecific absorption of molecules in the hydrophobic regions, so that the surfaces according to the invention have a very good signal-to-noise ratio. Furthermore, the surfaces according to the invention can be produced inexpensively.

According to a further aspect, the surfaces produced and / or modified according to the invention are provided as such.

Finally, the present invention relates to the use of the inventively modified surfaces in the context of analytical or diagnostic devices, as well as corresponding devices comprising the surfaces modified according to the invention.

The present invention will be explained in more detail with reference to a non-limiting embodiment.

embodiment

To produce the first hydrophobic layer, a linear polyacrylamide is applied to the carrier surface to be modified by spin coating and immobilized by irradiation with UV light in the presence of benzophenone as crosslinker. In a subsequent step, a linear polystyrene (likewise with benzophenone as crosslinker) is applied to this layer by means of spin coating and then selectively exposed, whereby the polystyrene layer in the region of the detection points is removed again.

Claims (8)

A method of modifying a support surface on which biomolecules are to be immobilized within discrete detection points or fields, comprising the following steps: (a) impinging the support surface with a hydrophobic layer; (b) creating one or more detection points or fields with hydrophilic properties by (i) the hydrophobic layer applied in step (a) is selectively removed in the case of an originally hydrophilic carrier surface, or (ii) the hydrophobic layer applied in step (a) is selectively removed in the case of an originally hydrophobic carrier surface and the originally hydrophobic carrier surface is converted into a hydrophilic surface. 2. The method according to claim 1, characterized in that in step (b) according to alternative (ii) to be carried out exposure and conversion by plasma etching. 3. The method according to claim 1 or 2, characterized in that the one or more in step (b) according to alternative (i) or (ii) created detection points or fields applied to a solution of crosslinkable and / or polymerisable components in a hydrophilic solvent and incubated such that the components in the solution in the detection point (s) form a crosslinked and / or polymerized layer having hydrophilic properties. 3. The method according to any one of claims 1 to 3, characterized in that the one or more created in step (b) detection points or fields hydrophilic conductive structures have, which are preferably formed as a star-shaped guide beams. 4. Analytical or diagnostic device having a surface which can be produced according to claim 3. 5. Analytical or diagnostic device, comprising a surface having both hydrophilic and hydrophobic regions, characterized in that the hydrophilic regions are formed as detection points or fields and have hydrophilic conductive structures, which are preferably configured as a star-shaped guide beams. 6. Analytical or diagnostic device according to claim 5, characterized in that the hydrophilic and hydrophobic areas differ from each other in terms of their color. 7. Use of a modified according to any one of claims 1 to 3 carrier surface for analytical or diagnostic purposes.

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